L-arginine and tetrahydrobiopterin protects against ischemia/reperfusion-induced endothelial dysfunction in patients with type 2 diabetes mellitus and coronary artery disease.

Diminished levels of L-arginine and endothelial nitric oxide synthase (eNOS) uncoupling through deficiency of tetrahydrobiopterin (BH(4)) may contribute to endothelial dysfunction. We investigated the effect of L-arginine and BH(4) administration on ischemia-reperfusion (I/R)-induced endothelial dysfunction in patients with type 2 diabetes and coronary artery disease (CAD). Forearm blood flow was measured by venous occlusion plethysmography in 12 patients with type 2 diabetes or impaired glucose tolerance and CAD. Forearm ischemia was induced for 20 min, followed by 60 min of reperfusion. The patients received a 15 min intra-brachial infusion of L-arginine (20 mg/min) and BH(4) (500 microg/min) or 0.9% saline starting at 15 min of ischemia on two separate study occasions. Compared with pre-ischemia the endothelium-dependent vasodilatation (EDV) induced by acetylcholine was significantly reduced at 15 and 30 min of reperfusion when saline was infused (P<0.001), but not following L-arginine and BH(4) infusion. EDV was also significantly less reduced at 15 and 30 min of reperfusion following L-arginine and BH(4) infusion, compared to saline infusion (P<0.02). Endothelium-independent vasodilatation (EIDV) induced by nitroprusside was unaffected by I/R. Venous total biopterin levels in the infused arm increased from 37+/-7 at baseline to 6644+/-1240 nmol/l during infusion of L-arginine and BH(4) (P<0.0001), whereas there was no difference in biopterin levels during saline infusion. In conclusion L-arginine and BH(4) supplementation reduces I/R-induced endothelial dysfunction, a finding which may represent a novel treatment strategy to limit I/R injury in patients with type 2 diabetes and CAD.

Severe endothelial dysfunction in the aorta of a mouse model of Fabry disease; partial prevention by N-butyldeoxynojirimycin treatment.

OBJECTIVE: Fabry disease results from alpha-gala-ctosidase A deficiency and is characterized by the lysosomal accumulation of globotriaosylceramide. Globotriaosylceramide storage predominantly affects endothelial cells, altering vascular wall morphology and vasomotor function. Our objective was to investigate aortic globotriaosylceramide levels, morphology and function in a mouse model of Fabry disease, and the effect of substrate reduction therapy, using the glycosphingolipid biosynthesis inhibitor N-butyldeoxynojirimycin. METHODS AND RESULTS: Mice used were C57BL/6J and alpha-galactosidase A knockout (Fabry). We show progressive accumulation of aortic globotriaosylceramide throughout the lifespan of untreated Fabry mice (55-fold elevation at 2 months increasing to 187-fold by 19 months), localized to endothelial and vascular smooth-muscle cells; there was no effect on vascular wall morphology in young Fabry mice. In old mice, storage resulted in intimal thickening. Endothelial function declined with age in Fabry mouse aorta. Aortae from N-butyldeoxynojirimycin-treated Fabry mice at 19 months of age had reduced endothelial globotriaosylceramide storage, fewer morphological abnormalities and less severe vasomotor dysfunction compared with untreated littermates. CONCLUSION: We provide evidence of a novel vascular phenotype in the Fabry mouse that has relevance to vascular disease in Fabry patients. N-Butyldeoxynojirimycin treatment partially prevented the phenotype in the Fabry mouse by reducing endothelial globotriaosylceramide storage.

Adenoviral gene transfer of nitric oxide synthase: high level expression in human vascular cells.

OBJECTIVES: Nitric oxide synthases (NOS) generate nitric oxide (NO), a second messenger with key regulatory roles. In the cardiovascular system, deficient endothelial NO production is an early, persistent feature of atherosclerosis and vascular injury. Accordingly, the NOS isoforms represent attractive targets for vascular gene therapy. We aimed to generate and evaluate an adenoviral vector for gene transfer of an NOS isoform to vascular cells. METHODS: We constructed a recombinant adenovirus, Ad.nNOS, for gene transfer of the neuronal isoform of NOS (nNOS) and characterized its expression in 293 cells, human vascular smooth muscle cells (hVSMC) and human umbilical vein endothelial cells (HUVEC). NOS expression was analyzed by Western immunoblotting, and NOS enzyme activity in response to receptor-dependent and receptor-independent agonists was determined by Griess assay or by NO chemiluminescence. RESULTS: Ad.nNOS-infected 293 cells expressed high levels of functional nNOS enzyme, even higher than in 293.NOS cells (a cell line that expresses supraphysiologic levels of nNOS). In hVSMC, nNOS activity reached levels 50% of those seen in 293.NOS cells. nNOS expression and activity in hVSMC increased linearly with titer of Ad.nNOS. NO production in hVSMC was stimulated both by calcium ionophore and by physiologic agonists such as acetylcholine or bradykinin. In HUVEC, endogenous NOS activity was significantly augmented by Ad.nNOS infection. Supplementation with the tetrahydrobiopterin precursor sepiapterin enhanced NOS activity in all cells. CONCLUSIONS: Ad.nNOS, a novel adenoviral vector for gene transfer of NOS, generates high-level nNOS expression in a variety of vascular cell types. nNOS activity in hVSMC is physiologically regulated and of a magnitude comparable to native eNOS activity in HUVEC. Our findings demonstrate Ad.nNOS to be a versatile and efficient tool for nNOS gene transfer, with widespread potential applications in cell culture and for gene therapy.